一种无需 PCR 的快速方法，用于在一个反应管中构建高度多样化的遗传文库。

A PCR-free rapid protocol for one-pot construction of highly diverse genetic libraries.

机构信息

Microbial Pathogenesis and Immunology Department, Texas A&M Health Science Center, Bryan, Texas, United States of America.

出版信息

PLoS One. 2022 Oct 31;17(10):e0276338. doi: 10.1371/journal.pone.0276338. eCollection 2022.

DOI:10.1371/journal.pone.0276338

PMID:36315516

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9621413/

Abstract

In vitro protein display methods can access extensive libraries (e.g., 1012-1014) and play an increasingly important role in protein engineering. However, the preparation of large libraries remains a laborious and time-consuming process. Here we report an efficient one-pot ligation & elongation (L&E) method for sizeable synthetic library preparation free of PCR amplification or any purification steps. As a proof of concept, we constructed an ankyrin repeat protein templated synthetic library with 1011 variants in 150 μL volume. The entire process from the oligos to DNA template ready for transcription is linearly scalable and took merely 90 minutes. We believe this L&E method can significantly simplify the preparation of synthetic libraries and accelerate in vitro protein display experiments.

摘要

体外蛋白质展示方法可以访问广泛的文库（例如，1012-1014），并在蛋白质工程中发挥越来越重要的作用。然而，大型文库的制备仍然是一个繁琐和耗时的过程。在这里，我们报告了一种有效的一锅法连接和延伸（L&E）方法，用于在无需 PCR 扩增或任何纯化步骤的情况下制备大量的合成文库。作为概念验证，我们构建了一个肌动蛋白重复蛋白模板的合成文库，其中包含 1011 个变体，体积为 150μL。从寡核苷酸到可用于转录的 DNA 模板的整个过程是线性可扩展的，仅需 90 分钟。我们相信这种 L&E 方法可以显著简化合成文库的制备，并加速体外蛋白质展示实验。